Hematopoietic stem cell transplantation (HSCT) is used as the major salvage strategy for patients with relapsed or refractory acute lymphoblastic leukemia (ALL), providing long-term disease control for some patients. Unfortunately, the majority of patients fail and relapse remains the most frequent cause of failure. The studies proposed here will utilize specimens obtained from patients enrolled on Children's Oncology Group ASCT0431. ASCT0431 is a nationwide randomized phase III study comparing two regimens (standard vs. sirolimus-based) designed to control graft vs. host disease (GVHD), and testing the hypothesis that the mTOR inhibitor sirolimus will control ALL at a point of minimal residual disease and thus decrease relapse risk and improve survival. Mechanisms of post-HSCT relapse fall into two broad categories: 1) the failure of anti-leukemic therapy to control disease;and 2) the ability of MRD to escape allogeneic immunosurveillance (the GVL effect). We hypothesize that sirolimus will improve the outcome of allogeneic HSCT for ALL by eliminating or suppressing residual ALL during the period post-HSCT when GVL may be absent due to immature immune reconstitution, thus providing adequate GVHD control without decreasing the GVL effect. A method to improve HSCT outcome in this fashion would be a major advance in transplantation and antileukemia therapy. These focused and integrated biological studies will assess the relative importance of sirolimus antileukemic effect and GVL directly in children treated for relapsed ALL with defined HSCT therapies and GVHD prophylaxis, and for whom blast samples from the initial relapse are available, in the critical setting of a Phase III trial. We will accomplish two Specific Aims: 1) Assess the impact of sirolimus on patient PBMC and on the ALL blasts. 2) Assess the impact of sirolimus-based GVHD prophylaxis on the GVL effect. The work is directly relevant to the mission of the agency because it seeks to improve transplant outcome, study the mechanisms of action of this agent in the context of a phase III study, and improve cure rates for patients with relapsed ALL. Lay Summary. We have very good treatments for the most common childhood leukemia, called ALL. However, if the disease comes back (relapse), most of the children will not survive. This research will look how a new drug used in bone marrow transplant, called sirolimus, may help prevent relapse in patients and study in the cells how it may act to prevent this problem.